Objective: Wound healing is a dynamic, interactive, and complex process that involves a series of events, including inflammation, migration, proliferation, granulation tissue formation, and matrix remodeling. Despite the high frequency of serious slow-healing wounds, there is still no adequate therapy. The aim of this study is to evaluate a new highly diluted acetylcholine (Ach) formulation obtained through a sequential kinetic activation (SKA) method applied to a wound healing in vivo model to verify the hypothesis that a low dose of Ach could be a more physiological stimulus for healing, by stimulating muscarinic and nicotinic receptors and their related intracellular pathways. Approach: Two different concentrations (10 fg/mL and 1 pg/mL) and two formulations (either kinetically or nonkinetically activated) of Ach were used to verify the wound healing process. Area closure, histological aspect, and nicotinic and muscarinic receptors, matrix metalloproteinase 9 (MMP-9), Nestin, and von Willebrand's factor have been assessed by Western blot or ELISA and compared to 147 ng/mL Ach, used as positive control. Moreover, the systemic effect through plasmatic radical oxygen species (ROS) production and Ach concentration has been evaluated. Results: Ach SKA 1 pg/mL revealed a significant capacity to restore the integrity of tissue compared to other formulation and this effect was more evident after a single administration. Innovation: Topical application on skin of Ach SKA 1 pg/mL accelerates wound closure stimulating non-neuronal cholinergic system. Conclusion: Our results demonstrate for the first time the importance in an in vivo model of highly diluted SKA Ach during wound healing, suggesting a potential use in skin disease.